The ability to silence genes via RNA interference (RNAi) was reported by Mello and Fire in 1998. See Fire et al., Nature (1998) 391:806-811. Since then, scientists have rushed to take advantage of the enormous therapeutic potential driven by targeted gene knockdown. This is evidenced by the fact that the first report of small interfering RNA (siRNA) mediated RNAi in human beings was reported only twelve years after the phenomenon was described in Caenorhabditis elegans. See Davis et al., Nature (2010) 464:1067-1070. The advantages of siRNA therapeutics include high target selectivity and specificity, and the potential to target pathways currently believed to be “undruggable” for the treatment of genetic diseases without effective therapy. siRNA therapeutics has shown promising results for the treatment of various diseases, such as hepatic carcinoma, hypercholesterolemia, refractory anemia, and familial amyloid neuropathy.
However, the efficient delivery of siRNA is still a challenge in the development of siRNA therapeutics. Due to issues associated with delivery efficiency and toxicity, the clinical use of siRNA requires safer and more effective delivery systems. It is understood that the development of genetic drugs is slowed by the inability to deliver nucleic acids effectively in vivo. When unprotected, genetic materials injected into the bloodstream can be degraded by deoxyribonucleases (DNAases) and ribonucleases (RNAases), or, if not degraded, the genetic materials can stimulate an immune response. See, e.g., Whitehead et al., Nature Reviews Drug Discovery (2009) 8:129-138; Robbins et al., Oligonucleotides (2009) 19:89-102. Intact siRNA must then enter the cytosol, where the antisense strand is incorporated into the RNA-induced silencing complex (RISC) (Whitehead et al., supra). The RISC associates with and degrades complementary mRNA sequences, thereby preventing translation of the target mRNA into protein, i.e., “silencing” the gene.
To overcome difficulties in delivery, polynucleotides have been complexed with a wide variety of delivery systems, including polymers, lipids, inorganic nanoparticles, and viruses. See, e.g., Peer et al., Nature Nanotechnology, (2007) 2:751-760. However, despite promising data from ongoing clinical trials for the treatment of respiratory syncytial virus infection and liver cancers (see, e.g., Zamora et al., Am. J. Respir. Crit. Care Med. (2011) 183:531-538), the clinical use of siRNA continues to require development of safer and more effective delivery systems. Toward this end, numerous lipid-like molecules have been developed including poly β-amino esters and amino alcohol lipids. See, e.g., International PCT Patent Application Publications, WO 2002/031025, WO 2004/106411, WO 2008/011561, WO 2007/143659, WO 2006/138380, WO 2010/053572, WO 2013/063468. Amino acid, peptide, polypeptide-lipids have also been studied for a variety of applications, including use as therapeutics, biosurfactants, and nucleotide delivery systems. See, e.g., Giuliani et al., Cellular and Molecular Life Sciences (2011) 68:2255-2266; Ikeda et al., Current Medicinal Chemistry (2007) 14: 111263-1275; Sen, Advances in Experimental Medicine and Biology (2010) 672:316-323; and Damen et al., Journal of Controlled Release (2010) 145:33-39.
Encapsulation of siRNAs within nanoparticles offers numerous delivery benefits, including protection from degradation by ubiquitous nucleases, passive and active targeting, and evasion of endosomal Toll-like receptors (1-9). To date, several polymeric, lipid, and dendritic nanoparticles have been developed for the encapsulation and delivery of siRNAs (1, 3, 5, 7-15). Despite the delivery successes met by some of these carriers, challenges to efficient delivery exist, including nanoparticle dissociation via serum proteins, cellular uptake, endosomal escape, and appropriate intracellular disassembly. To address some of these challenges, single parameter studies that evaluate the effect of chemical structure on a single biological property or on delivery performance have been reported (10-17). Furthermore, high-throughput synthetic methods have been exploited for the accelerated discovery of potent lipid nanoparticles (LNP) and evaluation of structure activity relationships (SARs) (16-20). In spite of these efforts, the relationships between physicochemical properties of nanoparticles and biological barriers, and that between biological barriers and gene silencing activity remain unclear. This lack of clarity has also resulted in poor in vitro-in vivo translation.
Therefore, there remains the need for new materials and systems for the delivery of siRNAs, other nucleic acids, and other agents to cells.